Novel human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/289,727, which was filed on May 9, 2001, and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosedpolynucleotides, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed polynucleotides, which can be used fordiagnosis, drug screening, clinical trial monitoring, the treatment ofdiseases and disorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Kinases mediate the phosphorylation of a wide variety of proteinsand compounds in the cell. Along with phosphatases, kinases are involvedin a range of regulatory pathways. Given the physiological importance ofkinases, they have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins andthe corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs) described for the first time herein sharestructural similarity with animal kinases, including, but not limitedto, adenylate kinases and phosphotransferases. Accordingly, thedescribed NHPs encode novel kinases having homologues and orthologsacross a range of phyla and species.

[0005] The novel human polynucleotides described herein encode openreading frames (ORFS) encoding proteins of 479 and 94 amino acids inlength (see, respectively, SEQ ID NOS:2 and 4).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHPs, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof at least one of the described NHPs. When the unique NHP sequencesdescribed in SEQ ID NOS:1-4 are “knocked-out” they provide a method ofidentifying phenotypic expression of the particular gene, as well as amethod of assigning function to previously unknown genes. In addition,animals in which the unique NHP sequences described in SEQ ID NOS:1-4are “knocked-out” provide a unique source in which to elicit antibodiesto homologous and orthologous proteins, which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses. To these ends, gene trappedknockout ES cells have been generated in murine homologs of thedescribed NHPs.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-4 are useful for the identification of protein coding sequences,and mapping a unique gene to a particular chromosome. These sequencesidentify biologically verified exon splice junctions, as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists of, NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of the novel humanORFs encoding the described novel human kinase proteins.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHPs described for the first time herein are novel proteinsthat are expressed in, inter alia, human cell lines and human fetalbrain, brain, pituitary, cerebellum, spinal cord, thymus, spleen, lymphnode, trachea, lung, kidney, fetal liver, prostate, testis, thyroid,adrenal gland, pancreas, salivary gland, stomach, colon, skeletalmuscle, heart, uterus, placenta, mammary gland, skin, esophagus,bladder, cervix, rectum, pericardium, hypothalamus, ovary, fetal kidney,fetal lung, gall bladder, tongue, aorta, 6-, 9-, and 12-week oldembryos, adenocarcinoma, osteosarcoma, embryonic carcinoma, umbilicalvein, and microvascular endothelial cells. Additional expression studiessuggest that the NHPs are also expressed in human bone marrow andadipose cells. The described sequences were compiled from sequencesavailable in GENBANK, and cDNAs generated from human mammary gland mRNAs(Edge Biosystems, Gaithersburg, Md.) that were identified using primersgenerated from human genomic DNA.

[0011] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described nucleotides, including thespecifically described NHPs, and the NHP products; (b) nucleotides thatencode one or more portions of a NHP that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences, including, but not limited to, the novel regions of anyactive domain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHPS, in which allor a part of at least one domain is deleted or altered, and thepolypeptide products specified by such nucleotide sequences, including,but not limited to, soluble proteins and peptides; (d) nucleotides thatencode chimeric fusion proteins containing all or a portion of a codingregion of a NHP, or one of its domains (e.g., a receptor/ligand bindingdomain, accessory protein/self-association domain, etc.) fused toanother peptide or polypeptide; or (e) therapeutic or diagnosticderivatives of the described polynucleotides, such as oligonucleotides,antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructscomprising a sequence first disclosed in the Sequence Listing.

[0012] As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2× SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet still encode a functionally equivalent NHPproduct. Functional equivalents of a NHP include naturally occurringNHPs present in other species, and mutant NHPs, whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,directed evolution as described in, for example, U.S. Pat. Nos.5,837,458 or 5,723,323 both of which are herein incorporated byreference). The invention also includes degenerate nucleic acid variantsof the disclosed NHP polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package, asdescribed herein, using default parameters).

[0014] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP-encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed herein. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80 bases long, or about34 to about 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0015] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a microarray or high-throughput“chip” format). Additionally, a series of NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS:1-4 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS:1-4, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon, are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405,the disclosures of which are herein incorporated by reference in theirentirety.

[0016] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-4 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. The probes typically comprise about 60nucleotides or can comprise about 25 nucleotides from the sequencesfirst disclosed in SEQ ID NOS:1-4.

[0017] For example, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, the described polynucleotide sequences shalltypically comprise at least about two or three distinct oligonucleotidesequences of at least about 8 nucleotides in length that are each firstdisclosed in the described Sequence Listing. Such oligonucleotidesequences can begin at any nucleotide present within a sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions, andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-4 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

[0019] Probes consisting of sequences first disclosed in SEQ ID NOS:1-4can also be used in the identification, selection, and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets, andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-4 can be utilized in microarrays, or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-4 in silico, andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0021] Thus the sequences first disclosed in SEQ ID NOS:1-4 can be usedto identify mutations associated with a particular disease, and also indiagnostic or prognostic assays.

[0022] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence, inconjunction with the presence of one or more specific oligonucleotidesequence(s) first disclosed in SEQ ID NOS:1-4. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

[0023] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6× SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP antisense molecules, useful, for example, inNHP gene regulation and/or as antisense primers in amplificationreactions of NHP nucleic acid sequences. With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0024] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0025] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0026] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0027] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0028] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

[0029] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (and periodic updates thereof), and Ausubel et al.,1989, supra.

[0030] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0031] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0032] Further, a NHP gene homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHP products disclosed herein. The templatefor the reaction may be total RNA, mRNA, genomic DNA and/or cDNAobtained by reverse transcription of mRNA prepared from, for example,human or non-human cell lines or tissue known to express, or suspectedof expressing, an allele of a NHP gene.

[0033] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0034] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to express, or suspected of expressing, a NHP gene). A reversetranscription (RT) reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see, e.g., Sambrooket al., 1989, supra.

[0035] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known to express, or suspected of expressing, a NHP, in anindividual putatively carrying a mutant NHP allele, and by extending thenew strand with reverse transcriptase. The second strand of the cDNA isthen synthesized using an oligonucleotide that hybridizes specificallyto the 5′ end of the normal sequence. Using these two primers, theproduct is then amplified via PCR, optionally cloned into a suitablevector, and subjected to DNA sequence analysis through methodswell-known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

[0036] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of carrying, or known to carry, amutant NHP allele (e.g., a person manifesting a NHP-associated phenotypesuch as, for example, behavioral disorders, immune disorders, obesity,high blood pressure, etc.), or a cDNA library can be constructed usingRNA from a tissue known to express, or suspected of expressing, a mutantNHP allele. A normal NHP gene, or any suitable fragment thereof, canthen be labeled and used as a probe to identify the corresponding mutantNHP allele in such libraries. Clones containing mutant NHP sequences canthen be purified and subjected to sequence analysis according to methodswell-known to those skilled in the art.

[0037] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.).

[0038] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to a NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0039] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721, 5,837,458, 6,117,679, and 5,723,323, which areherein incorporated by reference in their entirety.

[0040] The invention also encompasses: (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators, and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0041] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins (although processed forms or fragments can be secreted ormembrane associated), expression systems can be engineered that producesoluble derivatives of a NHP (corresponding to a NHP extracellularand/or intracellular domains, or truncated polypeptides lacking one ormore hydrophobic domains) and/or NHP fusion protein products (especiallyNHP-Ig fusion proteins, i.e., fusions of a NHP domain to an IgFc), NHPantibodies, and anti-idiotypic antibodies (including Fab fragments),that can be used in therapeutic applications. Preferably, the aboveexpression systems are engineered to allow the desired peptide orpolypeptide to be recovered from the culture media.

[0042] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0043] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs, or inappropriately expressed NHPs, for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

[0044] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway), can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of a soluble NHP, a NHP-IgFc fusion protein, or ananti-idiotypic antibody (or its Fab) that mimics the NHP, could activateor effectively antagonize an endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules, can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

[0045] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHP Sequences

[0046] The cDNA sequences and corresponding deduced amino acid sequencesof the described NHPs are presented in the Sequence Listing.

[0047] Expression analysis has provided evidence that the described NHPscan be expressed in a relatively wide range of human tissues. Inaddition to adenylate kinases, the described NHPs also share significantsimilarity to a range of additional kinase families, including kinasesassociated with signal transduction, from a variety of phyla andspecies.

[0048] A number of polymorphisms were identified during the sequencingof the NHPS, including: a G/C polymorphism at the region represented bynucleotide position number 427 of, for example, SEQ ID NO:1, which canresult in a glu or gin being present at corresponding amino acid (aa)position 143 of, for example, SEQ ID NO:2; and a C/G polymorphism at theregion represented by nucleotide position number 483 of, for example,SEQ ID NO:1, both of which result in a val being present atcorresponding aa position 161 of, for example, SEQ ID NO:2. The presentinvention contemplates NHPs comprising any or all of the abovepolymorphisms, as well as all combinations and permutations of thedescribed polymorphisms.

[0049] The gene encoding the described NHPs is apparently encoded onhuman chromosome 9 (see GENBANK accession no. AL161726). Accordingly,the described sequences are also useful for mapping and identifying thecoding regions of the human genome, and for defining exon splicejunctions.

[0050] Given the physiological importance of protein kinases, they havebeen subject to intense scrutiny, as exemplified and discussed in U.S.Pat. Nos. 5,756,289 and 5,817,479, and 6,340,583, herein incorporated byreference in their entirety, which additionally describe a variety ofuses and applications for the described NHPs.

[0051] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees, may be usedto generate NHP transgenic animals.

[0052] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0053] The present invention provides for transgenic animals that carrya NHP transgene in all their cells, as well as animals that carry atransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. A transgene may be integrated as asingle transgene, or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. A transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0054] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0055] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHP gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0056] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

[0057] The present invention also provides for “knock-in” animals.Knock-in animals are those in which a polynucleotide sequence (i.e., agene or a cDNA) that the animal does not naturally have in its genome isinserted in such a way that it is expressed. Examples include, but arenot limited to, a human gene or cDNA used to replace its murine orthologin a mouse, a murine cDNA used to replace the murine gene in a mouse,and a human gene or cDNA or murine cDNA that is tagged with a reporterconstruct used to replace the murine ortholog or gene in a mouse. Suchreplacements can occur at the locus of the murine ortholog or gene, orat another specific site. Such knock-in animals are useful for the invivo study, testing and validation of, intra alia, human drug targets,as well as for compounds that are directed at the same, and therapeuticproteins.

5.2 NHPS and NHP Polypeptides

[0058] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, and as reagents in assays for screening for compounds that canbe used as pharmaceutical reagents useful in the therapeutic treatmentof mental, biological, or medical disorders and diseases. Given thesimilarity information and expression data, the described NHPs can betargeted (by drugs, oligos, antibodies, etc.) in order to treat disease,or to therapeutically augment the efficacy of therapeutic agents.

[0059] The Sequence Listing discloses the amino acid sequences encodedby the described NHP-encoding polynucleotides. The NHPs displayinitiator methionines that are present in DNA sequence contextsconsistent with eucaryotic translation initiation sites.

[0060] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing, as well as analoguesand derivatives thereof. Further, corresponding NHP homologues fromother species are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described herein are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference), are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0061] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, the abilityto effect an identical or complementary downstream pathway, or a changein cellular metabolism (e.g., proteolytic activity, ion flux,phosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described herein, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0062] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHP peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of a NHP, but to assess biological activity, e.g., incertain drug screening assays.

[0063] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0064] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads, followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

[0065] In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreignpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. A NHP coding sequence can be cloned individually into anon-essential region (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a NHP coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted sequence isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

[0066] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, may be provided. Furthermore, theinitiation codon should be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al., 1987, Methods in Enzymol. 153:516-544).

[0067] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for the desired processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and in particular, humancell lines.

[0068] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described herein can be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci, which in turn can be cloned and expanded into cell lines.This method may advantageously be used to engineer cell lines thatexpress the NHP product. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the NHP product.

[0069] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0070] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.Another exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺-nitriloacetic acid-agarose columns, and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

[0071] Also encompassed by the present invention are fusion proteinsthat direct a NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching an appropriate signal sequence to a NHPwould also transport a NHP to a desired location within the cell.Alternatively targeting of a NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, N.Y., and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of NHPs to a target site ordesired organ, where they cross the cell membrane and/or the nucleuswhere the NHPs can exert their functional activity. This goal may beachieved by coupling of a NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences), to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization signals.

[0072] Additionally contemplated are oligopeptides that are modeled onan amino acid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

[0073] The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components that naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least about 75%, or at least about 90%,or at least about 99%, by weight. A substantially isolated or pureprotein or polypeptide may be obtained, for example, by extraction froma natural source, by expression of a recombinant nucleic acid encodingthe protein or polypeptide, or by chemically synthesizing the protein orpolypeptide.

[0074] Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants that accompany it inits natural state. Thus, a polypeptide that is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 Antibodies to NHP Products

[0075] Antibodies that specifically recognize one or more epitopes of aNHP, epitopes of conserved variants of a NHP, or peptide fragments of aNHP, are also encompassed by the invention. Such antibodies include, butare not limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0076] The antibodies of the invention can be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of a NHP. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes for the evaluation of the effect of test compounds onexpression and/or activity of a NHP expression product. Additionally,such antibodies can be used in conjunction with gene therapy to, forexample, evaluate normal and/or engineered NHP-expressing cells prior totheir introduction into a patient. Such antibodies may additionally beused in methods for the inhibition of abnormal NHP activity. Thus, suchantibodies may be utilized as a part of treatment methods.

[0077] For the production of antibodies, various host animals may beimmunized by injection with a NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), a truncated NHPpolypeptide (NHP in which one or more domains have been deleted),functional equivalents of a NHP or mutated variants of a NHP. Such hostanimals may include, but are not limited to, pigs, rabbits, mice, goats,and rats, to name but a few. Various adjuvants may be used to increasethe immunological response, depending on the host species, including,but not limited to, Freund's adjuvant (complete and incomplete), mineralsalts such as aluminum hydroxide or aluminum phosphate, chitosan,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Alternatively, the immune response could be enhanced bycombination and/or coupling with molecules such as keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin,or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0078] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof.The hybridomas producing the mAbs of this invention may be cultivated invitro or in vivo. Production of high titers of mAbs in vivo makes thisthe presently preferred method of production.

[0079] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,114,598,6,075,181 and 5,877,397 and their respective disclosures, which areherein incorporated by reference in their entirety. Also encompassed bythe present invention is the use of fully humanized monoclonalantibodies, as described in U.S. Pat. No. 6,150,584 and respectivedisclosures, which are herein incorporated by reference in theirentirety.

[0080] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0081] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of an antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0082] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHP and,therefore, bind, activate, or neutralize a NHP, NHP receptor, or NHPligand. Such anti-idiotypic antibodies, or Fab fragments of suchanti-idiotypes, can be used in therapeutic regimens involving aNHP-mediated pathway.

[0083] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen a NHP,and thus never been tolerized to a NHP) have a unique utility, as theycan be advantageously applied to the generation of antibodies againstthe disclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

[0084] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 4 1 1440 DNA homo sapiens 1 atggacgcca ctatcgcccc gcaccgtatcccccccgaga tgccccagta cggggaggag 60 aaccacatct tcgagttgat gcagaacatgctggagcaac tcctgatcca ccagcccgaa 120 gatcccatcc ccttcatgat ccagcacttgcatagagaca acgacaatgt gcccaggatt 180 gtaatattag gtccacccgc ctcagggaaaacaacaatag caatgtggct ctgcaaacat 240 ctgaacagca gtctcctcac cctggagaacctgatcttaa atgagttttc ctatacggcc 300 accgaagcca gaaggcttta tctgcaaaggaagacagttc ccagcgcgct gctcgtccag 360 ctgattcagg aacgcctggc tgaagaggattgcatcaagc agggctggat tctggatggc 420 atccctgaga cgcgtgagca ggctctgaggatccagaccc tggggatcac acccagacac 480 gtcattgtgc tgagtgctcc agacacggtcctgatcgaga gaaacttggg gaagagaatc 540 gaccctcaaa ctggagagat ttatcacaccacctttgact ggccacccga atctgaaatc 600 cagaaccgtc tcatggtgcc agaggacatctcagagctgg agacggctca gaaactgctg 660 gagtatcata ggaacatcgt cagggtcattccctcctacc ccaaaatcct caaagtcatc 720 agtgctgacc agccatgtgt ggacgtcttctaccaggctc tgacctatgt ccaaagcaac 780 catcgtacta atgccccgtt caccccgagggtgctgctgc tcgggcctgt gggcagtggg 840 aaaagtctgc aggccgccct cctggcccagaaatacaggc ttgtcaatgt ctgctgtggg 900 caactgctga aagaggctgt ggcagataggaccacgtttg gcgagctcat ccagcccttc 960 tttgaaaagg agatggcagt tcctgacagcctcctcatga aggtgctgag ccagcgcctg 1020 gaccagcagg actgcatcca gaaaggctgggtgctacacg gcgtcccgcg ggacctcgac 1080 caggcacacc tgctgaaccg cctgggctacaatcccaaca gggtgttttt cctgaatgtg 1140 ccatttgatt ccatcatgga gcggctgactctgagaagaa ttgatccagt cactggggaa 1200 aggtaccacc tcatgtacaa gccacctcccaccatggaga tccaggctcg cctcctgcag 1260 aacccaaagg atgctgaaga gcaggtcaagctgaaaatgg acctgttcta caggaactca 1320 gctgacttgg agcagttgta tgggtcggccatcaccctca atggggacca ggacccatac 1380 acagtcttcg aatacatcga gagtgggatcattaatcccc tgcccaagaa aatcccctga 1440 2 479 PRT homo sapiens 2 Met AspAla Thr Ile Ala Pro His Arg Ile Pro Pro Glu Met Pro Gln 1 5 10 15 TyrGly Glu Glu Asn His Ile Phe Glu Leu Met Gln Asn Met Leu Glu 20 25 30 GlnLeu Leu Ile His Gln Pro Glu Asp Pro Ile Pro Phe Met Ile Gln 35 40 45 HisLeu His Arg Asp Asn Asp Asn Val Pro Arg Ile Val Ile Leu Gly 50 55 60 ProPro Ala Ser Gly Lys Thr Thr Ile Ala Met Trp Leu Cys Lys His 65 70 75 80Leu Asn Ser Ser Leu Leu Thr Leu Glu Asn Leu Ile Leu Asn Glu Phe 85 90 95Ser Tyr Thr Ala Thr Glu Ala Arg Arg Leu Tyr Leu Gln Arg Lys Thr 100 105110 Val Pro Ser Ala Leu Leu Val Gln Leu Ile Gln Glu Arg Leu Ala Glu 115120 125 Glu Asp Cys Ile Lys Gln Gly Trp Ile Leu Asp Gly Ile Pro Glu Thr130 135 140 Arg Glu Gln Ala Leu Arg Ile Gln Thr Leu Gly Ile Thr Pro ArgHis 145 150 155 160 Val Ile Val Leu Ser Ala Pro Asp Thr Val Leu Ile GluArg Asn Leu 165 170 175 Gly Lys Arg Ile Asp Pro Gln Thr Gly Glu Ile TyrHis Thr Thr Phe 180 185 190 Asp Trp Pro Pro Glu Ser Glu Ile Gln Asn ArgLeu Met Val Pro Glu 195 200 205 Asp Ile Ser Glu Leu Glu Thr Ala Gln LysLeu Leu Glu Tyr His Arg 210 215 220 Asn Ile Val Arg Val Ile Pro Ser TyrPro Lys Ile Leu Lys Val Ile 225 230 235 240 Ser Ala Asp Gln Pro Cys ValAsp Val Phe Tyr Gln Ala Leu Thr Tyr 245 250 255 Val Gln Ser Asn His ArgThr Asn Ala Pro Phe Thr Pro Arg Val Leu 260 265 270 Leu Leu Gly Pro ValGly Ser Gly Lys Ser Leu Gln Ala Ala Leu Leu 275 280 285 Ala Gln Lys TyrArg Leu Val Asn Val Cys Cys Gly Gln Leu Leu Lys 290 295 300 Glu Ala ValAla Asp Arg Thr Thr Phe Gly Glu Leu Ile Gln Pro Phe 305 310 315 320 PheGlu Lys Glu Met Ala Val Pro Asp Ser Leu Leu Met Lys Val Leu 325 330 335Ser Gln Arg Leu Asp Gln Gln Asp Cys Ile Gln Lys Gly Trp Val Leu 340 345350 His Gly Val Pro Arg Asp Leu Asp Gln Ala His Leu Leu Asn Arg Leu 355360 365 Gly Tyr Asn Pro Asn Arg Val Phe Phe Leu Asn Val Pro Phe Asp Ser370 375 380 Ile Met Glu Arg Leu Thr Leu Arg Arg Ile Asp Pro Val Thr GlyGlu 385 390 395 400 Arg Tyr His Leu Met Tyr Lys Pro Pro Pro Thr Met GluIle Gln Ala 405 410 415 Arg Leu Leu Gln Asn Pro Lys Asp Ala Glu Glu GlnVal Lys Leu Lys 420 425 430 Met Asp Leu Phe Tyr Arg Asn Ser Ala Asp LeuGlu Gln Leu Tyr Gly 435 440 445 Ser Ala Ile Thr Leu Asn Gly Asp Gln AspPro Tyr Thr Val Phe Glu 450 455 460 Tyr Ile Glu Ser Gly Ile Ile Asn ProLeu Pro Lys Lys Ile Pro 465 470 475 3 285 DNA homo sapiens 3 atggagcggctgactctgag aagaattgat ccagtcactg gggaaaggta ccacctcatg 60 tacaagccacctcccaccat ggagatccag gctcgcctcc tgcagaaccc aaaggatgct 120 gaagagcaggtcaagctgaa aatggacctg ttctacagga actcagctga cttggagcag 180 ttgtatgggtcggccatcac cctcaatggg gaccaggacc catacacagt cttcgaatac 240 atcgagagtgggatcattaa tcccctgccc aagaaaatcc cctga 285 4 94 PRT homo sapiens 4 MetGlu Arg Leu Thr Leu Arg Arg Ile Asp Pro Val Thr Gly Glu Arg 1 5 10 15Tyr His Leu Met Tyr Lys Pro Pro Pro Thr Met Glu Ile Gln Ala Arg 20 25 30Leu Leu Gln Asn Pro Lys Asp Ala Glu Glu Gln Val Lys Leu Lys Met 35 40 45Asp Leu Phe Tyr Arg Asn Ser Ala Asp Leu Glu Gln Leu Tyr Gly Ser 50 55 60Ala Ile Thr Leu Asn Gly Asp Gln Asp Pro Tyr Thr Val Phe Glu Tyr 65 70 7580 Ile Glu Ser Gly Ile Ile Asn Pro Leu Pro Lys Lys Ile Pro 85 90

What is claimed is:
 1. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
 2. An isolatednucleic acid molecule comprising a nucleotide sequence that: (a) encodesthe amino acid sequence shown in SEQ ID NO:2; and (b) hybridizes understringent conditions to the nucleotide sequence of SEQ ID NO:1 or thecomplement thereof.
 3. An isolated nucleic acid molecule comprising anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:2.
 4. An isolated nucleic acid molecule comprising a nucleotidesequence encoding the amino acid sequence shown in SEQ ID NO:4.
 5. Asubstantially isolated prote in having the kinase activity of theprotein shown in SEQ ID NO:2 or SEQ ID NO:4, which is encoded by anucleotide sequence that hybridizes to SEQ ID NO:1 or SEQ ID NO:3 underhighly stringent conditions.